- Volume 137, Issue 4, 1991

Summary: Cells of Citrobacter freundii IFO 12681 contained glutathionylspermidine at a level of 2–3 nmol (g wet cells)−1. Three kinds of enzymes (E1, E2 and E3) catalysing the degradation of glutathionylspermidine were found in extracts of the bacterium. E1 was a spermidine dehydrogenase and was induced by spermidine. The enzyme hydrolysed glutathionylspermidine only in the presence of an electron acceptor. E2 and E3 were formed constitutively and required no cofactor for the degradation of glutathionylspermidine. E3 required at least two protein components for activity. The reaction products formed by E1, E2 and E3 differed from each other, but have not yet been identified. No activity generating glutathione and spermidine from glutathionylspermidine was detected in extracts of C. freundii.

Summary: An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 °C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5′ cap analogue 7-methylguanosine 5′-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 °C.

Summary: Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of β-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed β-oxidation, but the exact point(s) of difference have not yet been identified.

Summary: A biochemical analysis was undertaken of thermosensitive mutants of Bacillus subtilis 168 harbouring mutations in several tag genes, involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate), poly(groP). Incorporation of a pulse of [2-3H]glycerol into whole cells, following shift to the restrictive growth temperature, was used to assess synthesis of this polymer and to seek evidence of accumulation of a specific precursor. The rate of incorporation into poly(groP) was strongly decreased in all mutants; glycerol uptake was diminished by 80% or more for a strain harbouring mutation tagB1 (formerly tag-1) and one bearing tagD11 (formerly tag-11). The pool of CDP-glycerol (CDP-gro), a specific precursor of poly(groP), was increased, relative to the wild-type, for all mutations except tagD11, where the pool of CDP-gro was reduced. Cytoplasmic extracts, assayed at the permissive temperature for glycerol-3-phosphate cytidylyltransferase (gro-PCT), the enzyme synthesizing CDP-gro, revealed wild-type activities for all mutations except tagD11. Gro-PCT activity in the latter strain was 100-fold lower and, unlike that in all other mutant strains, highly thermolabile. This thermosensitivity suggests that tagD encodes gro-PCT. The identification, in a gene encoding a poly(groP)-specific enzyme, of a mutation conferring a thermosensitive growth phenotype renders explicit the conclusion that synthesis of this teichoic acid is essential for the growth of B. subtilis.

Summary: The subsite maps of two purified glucoamylases (P and S) from the fungus Hormoconis resinae were determined from kinetic data using sets of linear malto-and isomaltooligosaccharides as substrates. Glucoamylase P, which has an unusually high debranching (1,6-glucosidic) activity, showed a subsite map different from all known subsite maps of glucoamylases. The free energy of binding of maltooligosaccharides was negative at subsite 1, whereas all the others show a positive or zero energy at subsite 1. Inhibition of both glucoamylases P and S acting on either malto- or isomaltohexaose by gluconolactone [d-glucono-(1,5)-lactone] was investigated. Gluconolactone decreased the values of the maximum velocity, V, suggesting it can bind to subsite 1. The size of inhibition constants, identified as dissociation constants of gluconolactone from free enzyme, depended on whether the substrate was maltohexaose or isomaltohexaose. This suggests that gluconolactone has at least two binding sites, and that there are different subsites 1 (or 2) for 1,4- and 1,6-linked substrates. From previously reported results with other glucoamylases, an “induced fit” model was constructed for glucoamylases hydrolysing oligosaccharides.

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-K m isoenzyme I but the low-K m isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein (‘mannan’), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 °C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 °C but results in cell cycle arrest at 36 °C. These results are all consistent with the notion that in S. cerevisiae there are two genes (PGI1 and CDC30) which encode the low-K m and high-K m isoenzymes, respectively, of phosphoglucose isomerase and that the two gene products physically interact for activity.

Staphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL 10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1·0–1·5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35–40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50 % of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.

Summary: A derivative of Bacillus megaterium QM B1551 cured of all seven resident plasmids was mutated to Lac–2. Transposon Tn917 carrying lacZ and cat was then used to isolate four spo: :lacZ-cat fusion mutants by screening for colonies expressing the fusion in stationary phase. The sporulation frequencies of the mutants ranged from 10–7 to 10–2. Macrolide, lincosamide and streptogramin B resistance (MLSr) of the mutants cotransduced 100% with the sporulation defect and all four mutations mapped near trp by transduction in the order: trp–hisH–spo. All four mutants isolated were defective early in sporulation since β-galactosidase could be detected between zero and 2 h after the end of exponential growth. Electron microscopy of two of the mutants expressing the enzyme at t1–t2 revealed a defect prior to or just at the beginning of septum formation in one, and after completion of septum formation in the other. Little or no synthesis of dipicolinic acid, glucose dehydrogenase or alkaline phosphatase was detected in the mutants, but each was neutral protease positive. These results show that the mutations were in at least two early genes expressed before glucose dehydrogenase production. This study represents the first genetic characterization of sporulation mutants in B. megaterium and also demonstrates that gene fusion technology can be used in this species.

Three previously reported Tn5 mutants of Methylobacterium extorquens AM1, Cou-1, Cou-3 and Cou-6, which are able to grow on methylamine but not methanol, were characterized by biochemical analyses and complementation tests using two genomic libraries of M. extorquens AM1. We have designated the genes defective in these mutants as cou-1, cou-3 and cou-6 and mapped the site of Tn5 insertion in each. Biochemical results showed that two of these methanol oxidation (Mox) mutants, Cou-1 and Cou-3, are phenotypically similar to the previously identified MoxE class of mutant while Cou-6 resembled the MoxD class. Complementation tests and mapping the site of the Tn5 insertions indicated that cou-1 is another Mox gene linked to moxE and that cou-6 is linked to moxD. The Tn5 insertion in cou-3 mapped within the moxJ gene and therefore is the first detected mutation of the moxJ gene which was identified from expression studies. A MoxE mutant of M. extorquens AM1 was complemented by two different cosmid clones; one carried the moxE gene and the other contained two mox gene clusters, moxFJGI and moxAKLB. Hybridization experiments indicated that the moxE gene was not present on this latter clone, and it must therefore encode a gene capable of suppressing a MoxE mutation.

Summary: Insertional mutagenesis has revealed that a 22 kbp segment from the hisA region of the Bacillus subtilis 168 chromosome (310° on the genetic map) contains at least six independent transcription units, all apparently devoted to production of cell envelope components. Genes concerned with synthesis of poly(glycerol phosphate), poly(groP), an essential cell wall polymer in B. subtilis 168, are organized in two divergently transcribed operons denoted tagABC and tagDEF. Nucleotide sequence analysis indicates that three of these six genes encode extremely basic polypeptides. The deduced products of the tagABC operon may be involved in poly(groP) assembly and export, whereas those of the tagDEF operon, which are very hydrophilic, are more likely to be implicated in poly(groP) precursor biosynthesis. The first gene of the tagDEF operon encodes glycerol-3-phosphate cytidylyltransferase ( Pooley et al., 1991 , Journal of General Microbiology 137, 921–928) and its deduced product has significant homology with cholinephosphate cytidylyltransferase from yeast. There is also substantial homology between the deduced products of tagB in the tagABC operon and tagF in the tagDEF operon.

Summary: The resistance spectrum to bacteriophase ø3T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-β-d-glucopyranosyl-N-acetylgalactosamine 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to ø3T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBS1 transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, wherease the majority mapped between gtaB and tagB1 (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate). The fomer mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 ( Estrela et al., 1986 , Journal of General Microbiology 132, 411–415), map in a lucus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosamine-containing teichoic acid. A possible biological role of this polymer is discussed.

Summary: Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (ld-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, IssB and IssD, were shown to induce, at the restrictive temperature, a deficiency in ld-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group IssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-ll-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group IssD entail a thermosensitive aspartokinase I. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase I, not in the carboxylase, and to belong to the dapG locus. dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in ld-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.

Summary: Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3·4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3·4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1·4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine?Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.

Summary: The Bradyrhizobium japonicum fumarase gene (fumC-like) was cloned and sequenced, and a fumC deletion mutant was constructed. This mutant had a Nod+ Fix+ phenotype in symbiosis with the host plant, soybean, and growth in minimal medium with fumarate as sole carbon source was also not affected. The cloned B. japonicum fumC gene fully complemented an Escherichia coli Fum- mutant, strain JH400, for growth in minimal medium with fumarate. The predicted amino acid sequence of the FumC protein showed strong similarity to the E. coli FumC protein, Bacillus subtilis CitG protein, Saccharomyces cerevisiae Fum1 protein, and the mammalian fumarases. The B. japonicum FumC protein accounted for about 40% of the total fumarase activity in aerobically grown cells. The remaining 60% was ascribed to a temperature-labile fumarase. These data suggest that B. japonicum possesses two different fumarase isoenzymes, one of which is encoded by fumC. Besides E. coli, which has three fumarases, B. japonicum is thus the second bacterium for which there is genetic evidence for the existence of more than one fumarase.

Summary: Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12·6 kb consisting of the two contiguous HindIII fragments of 1·26 kb and 12·4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12·6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12·6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.